Nucleobase transporters (review)

Purines and pyrimidines play a key role in nucleic acid and nucleotide metabolism of all cells. In addition, they can be used as nitrogen sources in plants and many microorganisms. Transport of nucleobases across biological membranes is mediated by specific transmembrane transport proteins. Nucleobase transporters have been identified genetically and/or physiologically in bacteria, fungi, protozoa, algae, plants and mammals. A limited number of bacterial and fungal transporter genes have been cloned and analysed in great detail at the molecular level. Very recently, nucleobase transporters have been identified in plants. In other systems, with less accessible genetics, such as vertebrates and protozoa, no nucleobase transporter genes have been identified, and the transporters have been characterized and classified by physiological and biochemical approaches instead. In this review, it is shown that nucleobase transporters and similar sequences of unknown function present in databases constitute three basic families, which will be designated NAT, PRT and PUP. The first includes members from archea, eubacteria, fungi, plants and metazoa, the second is restricted to prokaryotes and fungi, and the last one is only found in plants. Interestingly, mammalian ascorbate transporters are homologous to NAT sequences. The function of different nucleobase transporters is also described, as is how their expression is regulated and what is currently known about their structure-function relationships. Common features emerging from these studies are expected to prove critical in understanding what governs nucleobase transporter specificity and in selecting proper model microbial systems for cloning and studying plant, protozoan and mammalian nucleobase transporters of agricultural, pharmacological and medical importance.     

Nucleobase transporters

Purines and pyrimidines play a key role in nucleic acid and nucleotide metabolism of all cells. In addition, they can be used as nitrogen sources in plants and many microorganisms. Transport of nucleobases across biological membranes is mediated by specific transmembrane transport proteins. Nucleobase transporters have been identified genetically and/or physiologically in bacteria, fungi, protozoa, algae, plants and mammals. A limited number of bacterial and fungal transporter genes have been cloned and analysed in great detail at the molecular level. Very recently, nucleobase transporters have been identified in plants. In other systems, with less accessible genetics, such as vertebrates and protozoa, no nucleobase transporter genes have been identified, and the transporters have been characterized and classified by physiological and biochemical approaches instead. In this review, it is shown that nucleobase transporters and similar sequences of unknown function present in databases constitute three basic families, which will be designated NAT, PRT and PUP. The first includes members fromarchea, eubacteria, fungi, plants and metazoa, the second is restricted to prokaryotes and fungi, and the last one is only found in plants. Interestingly, mammalian ascorbate transporters are homologous to NAT sequences. The function of different nucleobase transporters is also described, as is how their expression is regulated and what is currently known about their structure-function relationships. Common features emerging from these studies are expected to prove critical in understanding what governs nucleobase transporter specificity and in selecting proper model microbial systems for cloning and studying plant, protozoan and mammalian nucleobase transporters of agricultural, pharmacological and medical importance.     

Cryptic purine transporters in Aspergillus nidulans reveal the role of specific residues in the evolution of specificity in the NCS1 family

NCS1 proteins are H⁺ or Na⁺ symporters responsible for the uptake of purines, pyrimidines or related metabolites in bacteria, fungi and some plants. Fungal NCS1 are classified into two evolutionary and structurally distinct subfamilies, known as Fur‐ and Fcy‐like transporters. These subfamilies have expanded and functionally diversified by gene duplications. The Fur subfamily of the model fungus Aspergillus nidulans includes both major and cryptic transporters specific for uracil, 5‐fluorouracil, allantoin or/and uric acid. Here we functionally analyse all four A. nidulans Fcy transporters (FcyA, FcyC, FcyD and FcyE) with previously unknown function. Our analysis shows that FcyD is moderate‐affinity, low‐capacity, highly specific adenine transporter, whereas FcyE contributes to 8‐azaguanine uptake. Mutational analysis of FcyD, supported by homology modelling and substrate docking, shows that two variably conserved residues (Leu356 and Ser359) in transmembrane segment 8 (TMS8) are critical for transport kinetics and specificity differences among Fcy transporters, while two conserved residues (Phe167 and Ser171) in TMS3 are also important for function. Importantly, mutation S359N converts FcyD to a promiscuous nucleobase transporter capable of recognizing adenine, xanthine and several nucleobase analogues. Our results reveal the importance of specific residues in the functional evolution of NCS1 transporters.     

Characterization of novel Na+-dependent nucleobase transport systems at the blood-testis barrier

In the testis, nucleosides and nucleobases are important substrates of the salvage pathway for nucleotide biosynthesis, and one of the roles of Sertoli cells is to provide nutrients and metabolic precursors to spermatogenic cells located within the blood-testis barrier (BTB). We have already shown that concentrative and equilibrative nucleoside transporters are expressed and are functional in primary-cultured rat Sertoli cells as a BTB model, but little is known about nucleobase transport at the BTB or about the genes encoding specific nucleobase transporters in mammalian cells. In the present study, we examined the uptake of purine ([3H]guanine) and pyrimidine ([3H]uracil) nucleobases by primary-cultured rat Sertoli cells. The uptake of both nucleobases was time and concentration dependent. Kinetic analysis showed the involvement of three different transport systems in guanine uptake. In contrast, uracil uptake was mediated by a single Na+-dependent high-affinity transport system. Guanine uptake was inhibited by other purine nucleobases but not by pyrimidine nucleobases, whereas uracil uptake was inhibited only by pyrimidine nucleobases. In conclusion, it was suggested that there might be purine- or pyrimidine-selective nucleobase transporters in rat Sertoli cells.     

Systematic genetic dissection of PTS in Vibrio cholerae uncovers a novel glucose transporter and a limited role for PTS during infection of a mammalian host

A common mechanism for high affinity carbohydrate uptake in microbial species is the phosphoenolpyruvate‐dependent phosphotransferase system (PTS). This system consists of a shared component, EI, which is required for all PTS transport, and numerous carbohydrate uptake transporters. In Vibrio cholerae, there are 13 distinct PTS transporters. Due to genetic redundancy within this system, the carbohydrate specificity of each of these transporters is not currently defined. Here, using multiplex genome editing by natural transformation (MuGENT), we systematically dissect PTS transport in V. cholerae. Specifically, we generated a mutant strain that lacks all 13 PTS transporters, and from this strain, we created a panel of mutants where each expresses a single transporter. Using this panel, we have largely defined the carbohydrate specificities of each PTS transporter. In addition, this analysis uncovered a novel glucose transporter. We have further defined the mechanism of this transporter and characterized its regulation. Using our 13 PTS transporter mutant, we also provide the first clear evidence that carbohydrate transport by the PTS is not essential during infection in an infant mouse model of cholera. In summary, this study shows how multiplex genome editing can be used to rapidly dissect complex biological systems and genetic redundancy in microbial systems.     

Sugar transporters in plants and in their interactions with fungi

Sucrose and monosaccharide transporters mediate long distance transport of sugar from source to sink organs and constitute key components for carbon partitioning at the whole plant level and in interactions with fungi. Even if numerous families of plant sugar transporters are defined; efflux capacities, subcellular localization and association to membrane rafts have only been recently reported. On the fungal side, the investigation of sugar transport mechanisms in mutualistic and pathogenic interactions is now emerging. Here, we review the essential role of sugar transporters for distribution of carbohydrates inside plant cells, as well as for plant-fungal interaction functioning. Altogether these data highlight the need for a better comprehension of the mechanisms underlying sugar exchanges between fungi and their host plants.     

Amino acid residues N450 and Q449 are critical for the uptake capacity and specificity of UapA,a prototype of a nucleobase-ascorbate transporter family

Specific carrier-mediated transport of purine and pyrimidine nucleobases across cell membranes is a basic biological process in both prokaryotes and eukaryotes. Recent in silico analysis has shown that the Aspergillus nidulans (UapA, UapC) and bacterial (PbuX, UraA, PyrP) nucleobase transporters, and a group of mammalian L-ascorbic acid transporters (SVCT1 and SVCT2), constitute a unique protein family which includes putative homologues from archea, bacteria, plants and metazoans. The construction and functional analysis of chimeric purine transporters (UapA-U apC) and UapA-specific missense mutations in A. nidulans has previously shown that the region including amino acid residues 378-446 in UapA is critical for purine recognition and transport. Here, we extend our studies on UapA structure-function relationships by studying missense mutations constructed within a `signature' sequence motif [(F/Y/S)X(Q/E/P)N XGXXXXT(K/R/G)] which is conserved in the putative functional region of all members of the nucleobase/ascorbate transporter family. Residues Q449 and N450 were found to be critical for purine recognition and transport. The results suggest that these residues might directly or indirectly be involved in specific interactions with the purine ring. In particular, interaction of residue 449 with C-2 groups of purines might act as a critical molecular filter involved in the selection of transported substrates. The present and previous mutagenic analyses in UapA suggest that specific polar or charged amino acid residues on either side of an amphipathic a-helical transmembrane segment are critical for purine binding and transport.     

Amino acid residues N450 and Q449 are critical for the uptake capacity and specificity of UapA, a prototype of a nucleobase-ascorbate transporter family

Specific carrier-mediated transport of purine and pyrimidine nucleobases across cell membranes is a basic biological process in both prokaryotes and eukaryotes. Recent in silico analysis has shown that the Aspergillus nidulans (UapA, UapC) and bacterial (PbuX, UraA, PyrP) nucleobase transporters, and a group of mammalian L-ascorbic acid transporters (SVCT1 and SVCT2), constitute a unique protein family which includes putative homologues from archea, bacteria, plants and metazoans. The construction and functional analysis of chimeric purine transporters (UapA-UapC) and UapA-specific missense mutations in A. nidulans has previously shown that the region including amino acid residues 378-446 in UapA is critical for purine recognition and transport. Here, we extend our studies on UapA structure-function relationships by studying missense mutations constructed within a 'signature' sequence motif [(F/Y/S)X(Q/E/P)NXGXXXXT(K/R/G)] which is conserved in the putative functional region of all members of the nucleobase/ascorbate transporter family. Residues Q449 and N450 were found to be critical for purine recognition and transport. The results suggest that these residues might directly or indirectly be involved in specific interactions with the purine ring. In particular, interaction of residue 449 with C-2 groups of purines might act as a critical molecular filter involved in the selection of transported substrates. The present and previous mutagenic analyses in UapA suggest that specific polar or charged amino acid residues on either side of an amphipathic alpha-helical transmembrane segment are critical for purine binding and transport.     

Uniport,Not Proton-Symport,in a Non-Mammalian SLC23 Transporter

SLC23 family members are transporters of either nucleobases or ascorbate. While the mammalian SLC23 ascorbate transporters are sodium-coupled, the non-mammalian nucleobase transporters have been proposed, but not formally shown, to be proton-coupled symporters. This assignment is exclusively based on in vivo transport assays using protonophores. Here, by establishing the first in vitro transport assay for this protein family, we demonstrate that a representative member of the SLC23 nucleobase transporters operates as a uniporter instead. We explain these conflicting assignments by identifying a critical role of uracil phosphoribosyltransferase, the enzyme converting uracil to UMP, in driving uracil uptake in vivo. Detailed characterization of uracil phosphoribosyltransferase reveals that the sharp reduction of uracil uptake in whole cells in presence of protonophores is caused by acidification-induced enzyme inactivation. The SLC23 family therefore consists of both uniporters and symporters in line with the structurally related SLC4 and SLC26 families that have previously been demonstrated to accommodate both transport modes as well.     

AtAzg1 and AtAzg2 comprise a novel family of purine transporters in Arabidopsis

In plants, nucleobase biochemistry is highly compartmented relying upon a well-regulated and selective membrane transport system. In Arabidopsis two proteins, AtAzg1 and AtAzg2, show substantial amino acid sequence similarity to the adenine-guanine-hypoxanthine transporter AzgA of Aspergillus nidulans. Analysis of single and double mutant lines harboring T-DNA insertion alleles AtAzg1-1 and AtAzg2-1 reveal a marked resistance to growth in the presence of 8-azaadenine and 8-azaguanine but not to other toxic nucleobase analogues. Conversely, yeast strains expressing AtAzg1 and AtAzg2 gain heightened sensitivity to growth on 8-azaadenine and 8-azaguanine. Radio-labeled purine uptake experiments in yeast and in planta confirm the function of AtAzg1 and AtAzg2 as plant adenine-guanine transporters.     

Membrane transport metabolons

In this review evidence from a wide variety of biological systems is presented for the genetic, functional, and likely physical association of membrane transporters and the enzymes that metabolize the transported substrates. This evidence supports the hypothesis that the dynamic association of transporters and enzymes creates functional membrane transport metabolons that channel substrates typically obtained from the extracellular compartment directly into their cellular metabolism. The immediate modification of substrates on the inner surface of the membrane prevents back-flux through facilitated transporters, increasing the efficiency of transport. In some cases products of the enzymes are themselves substrates for the transporters that efflux the products in an exchange or antiport mechanism. Regulation of the binding of enzymes to transporters and their mutual activities may play a role in modulating flux through transporters and entry of substrates into metabolic pathways. Examples showing the physical association of transporters and enzymes are provided, but available structural data is sparse. Genetic and functional linkages between membrane transporters and enzymes were revealed by an analysis of Escherichia coli operons encoding polycistronic mRNAs and provide a list of predicted interactions ripe for further structural studies. This article supports the view that membrane transport metabolons are important throughout Nature in organisms ranging from bacteria to humans.     

Membrane transport metabolons

In this review evidence from a wide variety of biological systems is presented for the genetic, functional, and likely physical association of membrane transporters and the enzymes that metabolize the transported substrates. This evidence supports the hypothesis that the dynamic association of transporters and enzymes creates functional membrane transport metabolons that channel substrates typically obtained from the extracellular compartment directly into their cellular metabolism. The immediate modification of substrates on the inner surface of the membrane prevents back-flux through facilitated transporters, increasing the efficiency of transport. In some cases products of the enzymes are themselves substrates for the transporters that efflux the products in an exchange or antiport mechanism. Regulation of the binding of enzymes to transporters and their mutual activities may play a role in modulating flux through transporters and entry of substrates into metabolic pathways. Examples showing the physical association of transporters and enzymes are provided, but available structural data is sparse. Genetic and functional linkages between membrane transporters and enzymes were revealed by an analysis of Escherichia coli operons encoding polycistronic mRNAs and provide a list of predicted interactions ripe for further structural studies. This article supports the view that membrane transport metabolons are important throughout Nature in organisms ranging from bacteria to humans.     

ABC proteins in yeast and fungal pathogens

All fungal genomes harbour numerous ABC (ATP-binding cassette) proteins located in various cellular compartments such as the plasma membrane, vacuoles, peroxisomes and mitochondria. Most of them have initially been discovered through their ability to confer resistance to a multitude of drugs, a phenomenon called PDR (pleiotropic drug resistance) or MDR (multidrug resistance). Studying the mechanisms underlying PDR/MDR in yeast is of importance in two ways: first, ABC proteins can confer drug resistance on pathogenic fungi such as Candida spp., Aspergillus spp. or Cryptococcus neoformans; secondly, the well-established genetic, biochemical and cell biological tractability of Saccharomyces cerevisiae makes it an ideal tool to study basic mechanisms of drug transport by ABC proteins. In the past, knowledge from yeast has complemented work on human ABC transporters involved in anticancer drug resistance or genetic diseases. Interestingly, increasing evidence available from yeast and other organisms suggests that ABC proteins play a physiological role in membrane homoeostasis and lipid distribution, although this is being intensely debated in the literature.     

微生物有机酸转运蛋白的研究进展

转运蛋白是一类膜蛋白,可介导生物膜内外化学物质的跨膜转运及信号交换。有机酸转运蛋白在微生物有机酸代谢的跨膜转运过程中发挥重要作用,根据转运蛋白有机酸转运的方向不同可以分为摄取转运蛋白和外排转运蛋白。在微生物代谢中,有些有机酸可以作为能源直接参与体内代谢,有些是能量转换过程中的重要中间产物;摄取转运蛋白的过表达,可以促进微生物细胞获取能源物质,高效的生产目标产物;有机酸摄取转运蛋白敲除或外排转运蛋白表达,有利于底盘细胞外排更多目标产物,进而促进有机酸的生物合成。研究有机酸转运蛋白的结构和功能,有助于解析微生物细胞有机酸生物合成及利用的机制,对于提高工业微生物对有机酸的利用及生物合成具有重要作用。本文综述了微生物有机酸转运蛋白分类和结构、转运方式和转运功能等方面,重点综述了转运蛋白在有机酸生产中的应用,为工业微生物有机酸的高效生物合成及未来发展提供参考。     

Approaches for refining heterologous protein production in filamentous fungi

Fungi combine the advantages of a microbial system such as a simple fermentability with the capability of secreting proteins that are modified according to a general eukaryotic scheme. Filamentous fungi such as Aspergillus niger efficiently secrete genuine proteins but the secretion of recombinant proteins turned out be a difficult task. Aspergillus niger is an attractive organism because of its high secretion capacity and is frequently used as a model organism. Whereas high production yields can be obtained when homologous proteins are expressed, much lower amounts are obtained with the production of heterologous proteins. To fully exploit the potential of filamentous fungi, understanding of the molecular genetics, their physiology, and the glycosylation metabolism has to be investigated and clarified in more detail. This review summarizes recent developments in heterologous protein production by filamentous fungi and also generalizes the possibilities of improving the protein production by various genetic and bioprocessing approaches, thereby easing recognition of filamentous fungi as a relevant and reliable expression platform.     

Plant ABC transporters

The ATP binding cassette (ABC) superfamily is a large, ubiquitous and diverse group of proteins, most of which mediate transport across biological membranes. ABC transporters have been shown to function not only as ATP-dependent pumps, but also as ion channels and channel regulators. Whilst members of this gene family have been extensively characterised in mammalian and microbial systems, the study of plant ABC transporters is a relatively new field of investigation. Sequences of over 20 plant ABC proteins have been published and include homologues of P-glycoprotein, MRP, PDR5 and organellar transporters. At present, functions have been assigned to a small proportion of these genes and only the MRP subclass has been extensively characterised. This review aims to summarise literature relevant to the study of plant ABC transporters, to review methods of cloning, to discuss the utility of yeast and mammalian systems as models and to speculate on possible roles of uncharacterised ABC transporters in plants.     

Comparative and functional genomic analysis of prokaryotic nickel and cobalt uptake transporters: evidence for a novel group of ATP-binding cassette transporters

The transition metals nickel and cobalt, essential components of many enzymes, are taken up by specific transport systems of several different types. We integrated in silico and in vivo methods for the analysis of various protein families containing both nickel and cobalt transport systems in prokaryotes. For functional annotation of genes, we used two comparative genomic approaches: identification of regulatory signals and analysis of the genomic positions of genes encoding candidate nickel/cobalt transporters. The nickel-responsive repressor NikR regulates many nickel uptake systems, though the NikR-binding signal is divergent in various taxonomic groups of bacteria and archaea. B(12) riboswitches regulate most of the candidate cobalt transporters in bacteria. The nickel/cobalt transporter genes are often colocalized with genes for nickel-dependent or coenzyme B(12) biosynthesis enzymes. Nickel/cobalt transporters of different families, including the previously known NiCoT, UreH, and HupE/UreJ families of secondary systems and the NikABCDE ABC-type transporters, showed a mosaic distribution in prokaryotic genomes. In silico analyses identified CbiMNQO and NikMNQO as the most widespread groups of microbial transporters for cobalt and nickel ions. These unusual uptake systems contain an ABC protein (CbiO or NikO) but lack an extracytoplasmic solute-binding protein. Experimental analysis confirmed metal transport activity for three members of this family and demonstrated significant activity for a basic module (CbiMN) of the Salmonella enterica serovar Typhimurium transporter.     

ABC transporters: bacterial exporters.

The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain. ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters. We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems. The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates. We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates. Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family. The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti. Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches. One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides. Differences in substrate specificity do not correlate with evolutionary relatedness. A complete survey of the known and putative bacterial ABC exporters is included at the end of the review.